Nocturnal Salivary Cortisol Research Articles

Nocturnal Salivary Cortisol Is an Accurate Non-Invasive Test to Assess Endogenous Hypercortisolism in Children with Obesity and a Clinical Phenotype Suspicious for Cushing's Syndrome.

Cushing's syndrome (CS) constitutes one of the most challenging diagnostic assessments for paediatric endocrinologists. The clinical presentation of some children with exogenous obesity overlaps with those observed in hypercortisolism states. Accurate, non-invasive first-line tests are necessary to avoid false-positive results in the obese. We aimed to evaluate the diagnostic accuracy of salivary cortisol to assess endogenous hypercortisolism in children with obesity and clinical overlapping signs of CS. Case-control study that included children aged 2-18 years, BMI-SDS ≥2.0 and a follow-up >2 years. Patients were assigned to three categories: group A, features strongly indicative of paediatric CS (growth failure combined with increasing weight); group B, features suggestive of CS (e.g., moon face and striae); and group C, less specific features overlapping with CS (e.g., hypertension, hirsutism, insulin resistance). Children in categories A and B formed the control group. Ten patients with confirmed CS were the case group. All children collected saliva samples on the same day in the morning between 7 and 8:00 a.m. (morning salivary cortisol: mSC) and at 11 p.m. (nocturnal salivary cortisol: nSC). The mSC and nSC results were used to calculate the percentage decrease of cortisol at night (%D). Main outcomes by receiver operating characteristic for nSC and the %D were sensitivity, specificity, positive (P) and negative (N) predictive values (PV) and their corresponding 95% CI. Salivary cortisol was measured by electrochemiluminescence assay (lower limit of quantification: 2.0 nmol/L). 75/112 children met the inclusion criteria, whereas 22/75 children were eligible for the control group. Only controls decreased nSC (median and interquartile range: 2.0 [2.0-2.5] nmol/L) compared to mSC (6.9 [4.8-10.4] nmol/L), p < 0.0001. A cut-off for nSC ≥8 nmol/L confirmed CS within a sensitivity: 1.0 (0.69-1.0), specificity: 1.0 (0.85-1.0), PPV: 1.0 (0.69-0.99), and NPV: 1.0(0.85-0.99), achieving a diagnostic efficiency of 100%. The cut-off obtained for %D was 50%. No child with CS had a %D ≥50%, but 6/22 children in the control group had a %D below the cut-off, resulting in a lower overall diagnostic accuracy of 81% compared to nSC. Salivary cortisol at 11 p.m. is an accurate, feasible, and non-invasive first-line test to assess endogenous hypercortisolism in children with obesity and clinical suspicion of CS. The nSC was also useful in showing that the circadian rhythm of cortisol was preserved in children with exogenous obesity. In patients with nSC ≥8.0 nmol/L, other biochemical assessments and imaging studies are needed to further confirm the aetiology.

SAT324 Pulmonary Embolism Before And After Transsphenoidal Surgery In Cushing's Disease: Case Report.

Abstract Disclosure: A.B. Figueiredo: None. C.A. Baptista: None. C.R. Xavier: None. A.K. Nascimento: None. R.L. Dolci: None. A.L. dos Santos: None. J. Ethel: None. C.T. da Silva: None. N.M. Scalissi: None. J.V. Lima: None. Introduction: Patients with Cushing's syndrome (CS) have a 4-fold increased risk of thrombosis, due to increased synthesis of Von Willebrand factor and cortisol-stimulated fibrinogen, as well as increased synthesis of type 1 plasminogen activator inhibitor. The first 4 weeks after surgery are the period most susceptible to thromboembolism due to the worsening of the coagulation profile associated with the sudden reduction in the level of cortisol and its anti-inflammatory activity, leading to a thrombotic and inflammatory state. Case report: A 28-year-old woman, evaluated for bariatric surgery, was admitted to the emergency department complaining of dyspnea and palpitations that had started the day before. Supplementary oxygen therapy was instituted, the electrocardiogram showed a S1Q3T3 pattern, and chest angiotomography identified a thrombus in the arch of the right pulmonary artery and another in the left pulmonary artery. Thrombolysis was performed in the emergency room, followed by anticoagulation with intravenous heparin, resulting in clinical stability. He had typical symptoms of hypercortisolism such as obesity, early-onset hypertension, hirsutism, amenorrhea, and proximal weakness. During preparation for bariatric surgery, CS was suspected, with suppression test with dexamethasone and nocturnal salivary cortisol confirming hypercortisolism, and peripheral test with DDAVP and pituitary magnetic resonance imaging (MRI) confirming Cushing's disease by 8x4 mm adenoma. It was opted for clinical treatment with ketoconazole and anticoagulation for 6 months with transsphenoidal surgery. In the immediate postoperative period, it was planned to keep the patient off anticoagulants for the shortest possible time, with enoxaparin being restarted after surgery. On the 5th postoperative day, laboratory tests revealed cortisol of 1.7 mcg/dL and ACTH of 6.3 pg/mL, suggesting remission of the disease and supplementation with prednisone. On the 8th postoperative day, the patient complained of sudden dyspnea and desaturation, which led to the investigation of a new episode of pulmonary embolism. which was confirmed by chest angiotomography. Thus, a new Pulmonary Embolism was confirmed, this time with the use of full dose enoxaparin. The patient was referred to the ICU, where he received oxygen support and heparin infusion, with gradual clinical improvement and later, with anticoagulation for Warfarin with an INR goal of 2.5-3.0 and after 22 days of surgery, the patient achieved clinical stability and was discharged from the hospital. Conclusion: This case highlights the association of CS and thromboembolic events, especially Pulmonary Embolism. Presentation: Saturday, June 17, 2023

Alterations in cortisol and interleukin-6 secretion in patients with COVID-19 suggestive of neuroendocrine-immune adaptations

PurposeThe beneficial effect of glucocorticoids in coronavirus disease (COVID-19) is established, but whether adrenal cortisol secretion is impaired in COVID-19 is not fully elucidated. In this case-control study, we investigated the diurnal free bioavailable salivary cortisol secretion in COVID-19 patients.MethodsFifty-two consecutive COVID-19 patients—before dexamethasone treatment in cases required—recruited between April 15 to June 15, 2021, (NCT04988269) at Laikon Athens University-Hospital, and 33 healthy age- and sex-matched controls were included. Diurnal salivary cortisol (8 a.m., 12, 6, and 10 p.m.), plasma adrenocorticotropin (ACTH) and aldosterone, and serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels were assessed. Diurnal salivary dehydroepiandrosterone (DHEA) and IL-6 were also assessed in subgroups of patients.ResultsMedian CRP and IL-6 measurements were about sixfold higher in patients than controls (both p < 0.001) Morning salivary cortisol levels did not differ between the two groups, but patients exhibited higher median levels of evening and nocturnal salivary cortisol compared to controls [0.391 (0.054, 0663) vs. 0.081 (0.054, 0.243) μg/dl, p < 0.001 and 0.183 (0.090, 0.834) vs. 0.054 (0.054, 0.332) μg/dl, p < 0.001, respectively], resulting in higher time-integrated area under the curve (AUC) (4.81 ± 2.46 vs. 2.75 ± 0.810, respectively, p < 0.001). Circulating ACTH, DHEA, and aldosterone levels were similar in patients and controls. Serum IL-6, but not ACTH levels, was strongly correlated with nocturnal cortisol salivary levels (ρ = 0.555, p < 0.001) in patients.ConclusionsIncreased evening and nocturnal but not morning cortisol secretion may occur in even clinically mild COVID-19. In the context of acute viral infection (COVID-19), IL-6 may partially replace ACTH as a stimulus of the glucocorticoid-secreting adrenal zona-fasciculata without influencing the secretion of DHEA and aldosterone.Clinical trial registrationhttps://clinicaltrials.gov/ct2/show/NCT04988269?term=yavropoulou&draw=2&rank=3 (NCT04988269).

MON-312 The Effects of Cabergoline in Pre-Surgical and Recurrence Periods of Cushing’s Disease Patients

BACKGROUND: Dopaminergic agonist cabergoline (CAB) has been used in pharmacological treatment of Cushing’s disease (CD). The effect is attributed to the frequent expression of subtype 2 dopamine receptor in corticotropic tumors. However, in vivo studies demonstrated normalization of urinary cortisol (UC) in about 30-40% of cases over the long term, mainly after surgical failure. Objective: To evaluate the effect of CAB as monotherapy in early preoperative period and on recurrence of Cushing’s disease. Methods. A single-center retrospective study was conducted in a tertiary referral center. Twenty-one patients with confirmed CD were included. Median age was 32 years (13-70), 86% female, 10 with microadenomas, 11 with macroadenomas. They were diagnosed from 1986 to 2016 and used CAB as monotherapy either in the preoperative period (n=7, CABi) or in recurrence, before any other treatment (n=14, CABr). It was considered ‘complete response’ a 24h-UC normalization and ‘partial response’ a 24h-UC reduction >50%. UC was obtained on the last follow-up evaluation. Normalization of nocturnal salivary cortisol (NSC) on CAB was evaluated in most cases, as well as the larger tumor diameter by pituitary MRI, before and after CAB treatment. Results: Complete response was achieved in 29% (6/21) of subjects after 14.9±16.4 months of treatment with a mean dose of 2.2±1.0 mg/wweek. Partial response occurred in 9.5% (2/21). NSC normalized in 35% (6/17) and no variation in tumor diameter before and after CAB use was observed (n=13): 6.8±6.8 vs. 7.2±7.1 mm, respectively. There was no normalization of UC in CABi at the end of the treatment whereas in CABr, 43% (6/14) of patients reached complete response. CABi group was treated for 4.7±1.9 months and CABr was treated for 20.1±18.1 months. Both groups were on similar doses of CAB (CABi 2.1±0.9 and CABr 2.3±1.1 mg/w). Interestingly, the difference between the groups’ complete response was evident early on 3 months of treatment: no cases in CABi vs. 60% (6/10) in CABr (p=0.035) despite a lower dose in CABr (1.0 vs. 1.5; p=0.008). Normalization of NSC occurred in 20% in CABi and 42% in CABr. Conclusion. Normalization of UC and NSC occurred in about 30% of total patients, mainly those who used CAB on recurrence of CD. Due to the small number of subjects in CABi group, the absence of hormone control in this group requires further investigation in order to verify the effectiveness of CAB as primary therapy or as preoperative treatment option.

Evaluation of the hypothalamic\u2013pituitary\u2013adrenal axis in a case series of familial partial lipodystrophy

BackgroundFamilial partial lipodystrophy (FPL) is a rare genetic disease characterized by body fat abnormalities that lead to insulin resistance (IR). Clinical conditions linked to milder IR, such as type 2 diabetes (T2D) and metabolic syndrome, are associated with abnormalities of the hypothalamic–pituitary–adrenal (HPA) axis, but little is known about its activity in FPL.MethodsPatients meeting the clinical criteria for FPL were subjected to anthropometric, biochemical and hormone analyses. A genetic study to identify mutations in the genes encoding peroxisome proliferator-activated receptor gamma (PPARγ) was performed. Polycystic ovary syndrome and hepatic steatosis were investigated, and the patient body compositions were analyzed via dual X-ray energy absorptiometry (DXA). The HPA axis was assessed via basal [cortisol, adrenocorticotrophic hormone (ACTH), cortisol binding globulin, nocturnal salivary cortisol and urinary free cortisol (UFC)] as well as dynamic suppression tests (cortisol post 0.5 mg and post 1 mg dexamethasone).ResultsSix patients (five female and one male) aged 17 to 42 years were included. In DXA analyses, the fat mass ratio between the trunk and lower limbs (FMR) was > 1.2 in all phenotypes. One patient had a confirmed mutation in the PPARγ gene: a novel heterozygous substitution of p. Arg 212 Trp (c.634C>T) at exon 5. HPA sensitivity to glucocorticoid feedback was preserved in all six patients, and a trend towards lower basal serum cortisol, serum ACTH and UFC values was observed.ConclusionsOur findings suggest that FPL is not associated with overt abnormalities in the HPA axis, despite a trend towards low-normal basal cortisol and ACTH values and lower UFC levels. These findings suggest that the extreme insulin resistance occurring in FPL may lead to a decrease in HPA axis activity without changing its sensitivity to glucocorticoid feedback, in contrast to the abnormalities in HPA axis function in T2D and common metabolic syndrome.

Negative correlation between tumour size and cortisol/ACTH ratios in patients with Cushing's disease harbouring microadenomas or macroadenomas.

Pituitary macroadenomas (MACs) represent 10-30% of Cushing's disease (CD) cases. The aim of this study was to report the clinical, laboratorial and imaging features and postsurgical outcomes of microadenoma (MIC) and MAC patients. Retrospective study with 317 CD patients (median 32years old, range 9-71years) admitted between 1990 and 2014, 74 (23.3%) of whom had MAC. Hirsutism, plethora facial, muscular weakness and muscular atrophy were more frequent in the MIC patients. Nephrolithiasis, osteopenia, hyperprolactinaemia and galactorrhoea were more prevalent in MAC patients. The morning serum cortisol (Fs), nocturnal salivary cortisol (NSC), nocturnal Fs (Fs 2400h), low- and high-dose dexamethasone suppression test results and CRH and desmopressin test results were similar between the subgroups. MIC patients showed higher urinary cortisol at 24h (UC), and MAC patients presented higher ACTH levels but lower Fs/ACTH, Fs 2400h/ACTH, NSC/ACTH and UC/ACTH ratios. There were negative correlations of tumour size with Fs/ACTH, Fs 2400h/ACTH, NSC/ACTH and UC/ACTH ratios. Overall, the postsurgical remission and recurrence rates were similar between MIC and MAC. However, patients in remission (MIC+MAC) showed smaller tumour diameters and a lower prevalence of invasion and extension on MRI. Despite exhibiting higher plasma ACTH levels, CD patients with MAC presented lower cortisol/ACTH ratios than did patients with MIC, with a negative correlation between tumour size and cortisol/ACTH ratios. The overall postsurgical remission and recurrence rates were similar between MIC and MAC patients, with those with larger and/or invasive tumours showing a lower remission rate.

Prevalence of subclinical hypercortisolism in type 2 diabetic patients from the Rio de Janeiro Type 2 Diabetes Cohort Study

AimsSubclinical hypercortisolism was reported to be more prevalent among diabetic, obese and hypertensive patients. Our primary aim was to investigate the prevalence of subclinical hypercortisolism in patients from the Rio de Janeiro Type 2 Diabetes (RIO-T2D) Cohort; and secondarily to assess its associated factors. MethodsFrom May 2013 to August 2014, 393 diabetic outpatients underwent overnight 1mg dexamethasone suppression test (DST). Patients with non-suppressive morning cortisol (≥1.8μg/dl) were further evaluated with nocturnal salivary cortisol, two readings >0.35μg/dl were considered confirmatory for subclinical hypercortisolism. ResultsOne-hundred twenty-eight patients (32.6%) failed to suppress morning cortisol, and in 33 patients (8.6%) subclinical hypercortisolism was confirmed. Independent correlates of a positive DST were older age (OR: 1.04; 95% CI: 1.01–1.07; p=0.007), number of anti-hypertensive drugs in use (OR: 1.26; 95% CI: 1.05–1.50; p=0.012), longer diabetes duration (OR: 1.03; 95% CI: 1.004–1.06; p=0.023), and presence of diabetic nephropathy (OR: 1.70; 95% CI: 1.01–2.87; p=0.047). Independent correlates of confirmed subclinical hypercortisolism were a greater number of anti-hypertensive medications (OR: 1.54; 95% CI: 1.14–2.06; p=0.004), shorter diabetes duration (OR: 0.92; 95% CI: 0.87–0.98; p=0.006), and increased aortic stiffness (OR: 2.81; 95% CI: 1.20–6.57; p=0.017); metformin use was protective (OR: 0.27; 95% CI: 0.10–0.73; p=0.010). ConclusionPatients with type 2 diabetes had a high prevalence of subclinical hypercortisolism, and its presence was associated with more severe hypertension and increased aortic stiffness.

Blunted nocturnal cortisol rise is associated with higher carotid artery intima-media thickness (CIMT) in overweight African American and Latino youth

Blunted diurnal cortisol variation has been associated with overt cardiovascular disease in adults. The relationship between the diurnal cortisol variation and subclinical atherosclerosis in youth has yet to be investigated. The objectives of this study were to (1) determine the relationship between overnight cortisol measures and CIMT in overweight and obese, African-American and Latino children; (2) assess ethnic differences in these relationships; and (3) explore whether overnight cortisol and CIMT relationships were independent of inflammatory markers, C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-∝ (TNF-∝). One hundred fifty-six overweight and obese African-American and Latino children (ages 8-17, 86 M/70 F, 55 African-American/101 Latino) underwent measures of CIMT by B-mode ultrasound, nocturnal cortisol rise (NCR = salivary cortisol rise from 2200 h to awakening at 0530 h), cortisol awakening response (CAR = salivary cortisol from time of awakening to 30 min later), fasting serum cortisol and overnight urinary free cortisol. Using linear regression, salivary cortisol(0530 h) and NCR were negatively associated with CIMT (β(standardized) = -0.215 and -0.220, p < 0.01) independent of age, height, percent body fat, ethnicity and systolic blood pressure. Nocturnal salivary cortisol(2200 h), morning serum cortisol, and overnight urinary free cortisol were not associated with CIMT. Using ANCOVA, participants with LOW NCR (NCR < 0.44 μg/dL, n = 52) had significantly greater CIMT than those with HIGH NCR (NCR ≥ 0.91 μg/dL, n = 52; 0.632 ± 0.008 vs. 0.603 ± 0.008 mm, p=0.01) after controlling for covariates. Ethnicity was independently associated with CIMT, whereby African-American children had greater CIMT than Latino children (-0.028 ± 0.009, p=0.006). The relationships between cortisol measures and CIMT did not differ between the two ethnic groups (all p(interaction) = 0.28-0.97). CRP, IL-6 and TNF-∝ were not associated with CIMT (p > 0.05). IL-6 was inversely related to NCR (r = -0.186, p = 0.03), but it did not explain the relationship between NCR and CIMT. Salivary cortisol(0530 h) and NCR, but not CAR, nocturnal salivary cortisol(2200 h), morning serum cortisol or overnight urinary free cortisol were associated with CIMT, independent of relevant covariates, including inflammatory factors. A low awakening salivary cortisol or a blunted NCR may be related to increased atherosclerosis risk in overweight and obese minority youth. These findings support adult studies suggesting flattened daytime diurnal cortisol variation impacts cardiovascular disease risk.

A Comparison of the Use of Urinary Cortisol to Creatinine Ratios and Nocturnal Salivary Cortisol in the Evaluation of Cyclicity in Patients with Cushing's Syndrome

Cyclical Cushing's syndrome is detected in our center by collecting sequential early morning urine (EMU) samples for cortisol to creatinine ratio over 28 d. The Endocrine Society suggests that nocturnal salivary cortisol (NSC) may be used to assess patients for cyclical Cushing's. However, there is only very limited evidence that it correlates with EMU testing or that it demonstrates cycling over 28 d. We sought to correlate NSC with EMU results collected the following morning and to determine whether NSC could be used to detect cyclical Cushing's. An observation study of 28-d collections for NSC and EMU was performed in a tertiary referral center over 1 yr. A 28-d collection of NSC and EMU was performed in 10 patients with confirmed or suspected Cushing's syndrome. The main outcome of the study was the correlation of salivary and urinary cortisol with graphical assessment of results for cycling. Eleven collections were performed. One patient with cyclical Cushing's completed the collection before and after cabergoline therapy. Two hundred seventy matched salivary and urinary results were correlated (r = 0.79; P < 0.001). In two patients with cyclical Cushing's, EMU and NSC followed a similar cyclical pattern. In one patient with recurrent cyclical Cushing's, cortisol was elevated in both saliva and urine but with more prominent cycles in saliva. NSC correlated well with EMU. NSC detected all cases of cyclical Cushing's. Therefore, NSC may prove to be an additional option or replacement for EMU in detecting cyclical Cushing's syndrome.

Day-to-day variation of late-night salivary cortisol in healthy voluntaries

Objectives The study examines the components of biological variation of nocturnal salivary cortisol in healthy subjects. Design and methods Eight repetitive measurements were performed in seven subjects during a 25-day time period (study A), and then, for comparison, two salivary specimens were taken during two consecutive days from 20 subjects (study B). Salivary cortisol was measured with the Salimetrics HS-Cortisol assay. Results Mean salivary cortisol (1.27 nmol/L), analytical variation (CV a = 15.4%), within-subject variation (CV i = 34.1%), between-subject variation (CV g = 35.3%), index of individuality (II = 1.06) and reference change value (RCV = 104%) were obtained for study A. Similar results were obtained from the set of samples of study B. Conclusion The study results show a medium degree of individuality for salivary cortisol. Both conventional reference values and comparison of serial results may be equally used for clinical interpretation. A change greater of 104% between two successive measurements should be considered significant.

Screening for Subclinical Cushing's Syndrome in Type 2 Diabetes Mellitus: Low False-Positive Rates with Nocturnal Salivary Cortisol

The diagnosis of subclinical Cushing's syndrome (SCS) is important, but its relative rarity amongst patients with common metabolic disorders requires a simple test with a low false-positive rate. Using nocturnal salivary cortisol (NSC), which we first validated in patients with suspected and proven Cushing's syndrome, we screened 106 overweight patients with type 2 diabetes mellitus, a group at high risk of SCS and nontumoral hypothalamic-pituitary-adrenal axis perturbations. Our hypothesis was that a lower false-positive rate with NSC was likely, compared with that reported with the dexamethasone suppression test (DST) (10-20%), currently the foundation of diagnosis of SCS. No participant had clinically apparent Cushing's syndrome. Three participants had an elevated NSC but further testing excluded SCS. In this study, NSC had a lower false-positive rate (3%) than previously reported for the DST. Given the reported excellent performance of NSC in detection of hypercortisolism, the low false-positive rate in SCS suggests NSC may be superior to the DST for SCS screening. The NSC and DST should be compared directly in metabolic disorder patients; although our data suggest the patient group will need to be substantially larger to definitively determine the optimal screening test.

Diagnóstico y diagnóstico diferencial del síndrome de Cushing

Because of the variability in the clinical expression of Cushing's syndrome, different biochemical tests and imaging techniques must be used for effective diagnosis and treatment. The clinical history is important to assess the general impact of hypercortisolism on organs and systems, as well as to guide suspicion toward more aggressive entities such as overt ectopic ACTH syndrome or to detect an iatrogenic etiology of Cushing's syndrome. The screening phase relies on tests that are sensitive but not specific, such as urinary free cortisol, nocturnal salivary cortisol and 1 mg dexamethasone suppression, which still require established assessment criteria. Confirmation can be obtained by repeating urinary cortisol, 2 mg/day dexamethasone suppression, both alone and combined with corticotropin releasing hormone (CRH), to exclude pseudoCushing states. ACTH dependency can be easily explored by ACTH measurements, but the differential diagnosis between pituitary and ectopic Cushing's disease is difficult and may require invasive procedures such as inferior petrosal sinus sampling. Sophisticated imaging and isotopic techniques play a significant role in locating the source of ACTH in ectopic syndromes but are not always effective. In general, biochemical and imaging tests should be combined in order to assess different mechanisms and perspectives of the syndrome. Rigorous methodology is essential to obtain accurate results, allowing a correct diagnosis and improving therapeutic performance in this devastating disease.

Limitations of nocturnal salivary cortisol and urine free cortisol in the diagnosis of mild Cushing's syndrome

Cushing's syndrome (CS) is difficult to diagnose due to its nonspecific presentation. Diagnostic tests like 24-h urine free cortisol (UFC) and the overnight 1 mg dexamethasone suppression test (DST) lack sufficient sensitivity and specificity. Measurement of nocturnal salivary cortisol (NSC) is an accurate and reproducible test with a high sensitivity for CS. However, its performance in mild CS has not been reported. We present 11 cases of CS with normal or mildly elevated UFC in whom NSC was helpful in making a diagnosis. All patients had at least one collection of 24-h UFC and NSC and eight had an overnight 1 mg DST. The number of NSC measurements per patient was determined by the clinical index of suspicion and the results of initial testing. Imaging studies included magnetic resonance imaging (MRI) of pituitary or computer tomography scan of abdomen. Only four out of eleven patients had elevations in UFC and none were >2 times the upper limit of normal. Seven out of eight had an abnormal DST. All patients had some elevated NSCs (14-100%). Out of eleven patients, six had an abnormality in the pituitary gland found by MRI and two out of eleven had adrenal masses. The remaining three had normal pituitary MRI but had inferior petrosal sinus (IPS) sampling indicating Cushing's disease. All patients had appropriate surgery, and histopathology of all except one was suggestive of either a cortisol-producing adrenal adenoma or an ACTH-secreting pituitary adenoma. Neither a normal UFC nor a normal NSC excludes mild CS. Multiple samples (urine/saliva) and DST are needed to make the diagnosis of mild CS.